Fatani, Abdulhamid; (2023) Gene therapy in combination with an approach targeting brain cancers. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background Glioblastoma multiforme (GBM) is a fast-growing and aggressive brain tumour classified as a grade IV astrocytoma. In spite of tremendous work to develop effective therapeutic strategies for GBM, including surgery, chemotherapy, radiation and combinations thereof, the prognosis is still poor, with a median survival time of about 12–18 months. The reasons for this include a combination of radioand chemo-resistance and the ability of malignant cells to spread broadly through normal brain tissue, rendering them difficult or impossible to surgically resect. Itch is an E3 ligase that is overexpressed in different cancers and has a critical role in inhibiting and degrading P73, a tumour suppressor gene. Targeting the ITCH gene is a promising therapeutic strategy for treating different types of cancer, including GBM, and for improving chemo-sensitivity. GBM can metastasize to reach intraocular tissues of the eye, causing a tumour called malignant optic nerve glioblastoma Methods We used glycol chitosan (GC60) with a molecular weight of 60 kDa to deliver plasmids with different reporters (luciferase and GFP) and therapeutic genes (siRNA anti-ITCH) in vitro and in vivo via noseto- brain administration. The complexation of GC60 and nucleic acids was assessed using different techniques, including dynamic light scattering, gel electrophoresis and transmission electron microscopy. Cell viability and apoptosis were evaluated in vitro by annexin V apoptosis assays and MTT assays and confirmed with flow cytometry and confocal microscopy. The levels of Itch and tumour suppressor gene P73 in vitro were measured using Western blot and PCR. Intranasal luciferase–DNA delivery to the brain was evaluated in BALB-C mice using an in vivo imaging system (IVIS) machine and bioluminescence assays of homogenized extracted brain tissues. The downregulatory effect of siRNA targeting ITCH alone or in combination with gemcitabine (chemotherapy) in CD-1 mice bearing GBM cells (U-87 MG-luc2) was assessed by PCR, Western blot, IVIS machine, survival rates and histopathology. Ocular gene delivery was assessed by IVIS and luminance measurement in rats’ homogenized tissues. Results In a dose-dependent manner, GC60 successfully delivered luciferase DNA to BALB-C mice brains via the nose-to-brain route of administration. Furthermore, siRNA anti-ITCH-loaded GC60 treatment potently reduced ITCH gene expression and increased the level of tumour suppressor gene P73 in vitro. Simultaneous ITCH inhibition greatly reduced GBM cell proliferation and induced apoptosis in vitro. Although there was little reduction in tumour growth with siRNA anti-ITCH alone in the IVIS machine, the combination of siRNA anti-ITCH-loaded GC60 and gemcitabine treatment significantly decreased tumour growth in CD-1 mice bearing bioluminescent U87 MG-Luc2 and decreased chemo-resistance to gemcitabine. A Kaplan-Meier assay revealed that the combination treatment of siRNA anti-ITCH levels and gemcitabine increased the survival of CD-1 nude mice bearing U87 MG-Luc2. Furthermore, the ocular gene delivery study demonstrated great gene expression in Sprague Dawley rats’ eyes. Conclusions Using GC60 to deliver therapeutic nucleic acids could represent a novel and effective approach for designing brain cancer gene therapies. Targeting the ITCH gene is a promising therapeutic strategy for inhibiting proliferation, inducing apoptosis in GBM and decreasing chemotherapy resistance.

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